Kiss1/Gpr54 Prevents Bone Loss through Src Dephosphorylation by Dusp18 in Osteoclasts

Osteoclasts were over-activated as we age, which leads to bone loss. Src-deficient mice lead to only one phenotype -severe osteopetrosis due to functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCR) have been targets for ∼35% of approved drugs. However, how Src kinase activity is negatively regulated by GPCRs remains largely elusive. Herein we report that Src is dephosphorylated at Tyr 416 by Dusp18 upon GPR54 activation by its natural ligand Kp-10. Mechanically, both active Src and the Dusp18 phosphatase are recruited by GPR54 through the proline/arginine-rich motif (PR motif) in the C terminus, which is dependent on the Gαq signal pathway. As such, Kiss1, Gpr54, Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss. Accordingly, Kp-10 abrogated bone loss by suppressing osteoclasts activity in vivo. Therefore, Kiss1/Gpr54 is a promising therapeutic strategy governing bone resorption through Src dephosphorylation by Dusp18.

Binding stoichiometry and structural model of the HIV-1 Rev/Importin beta complex

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein Importin beta (ImpB), but how Rev associates with ImpB is poorly understood. Here we report biochemical, biophysical and structural studies of the ImpB/Rev complex. Binding and mutagenesis data reveal that ImpB associates with two Rev monomers through independent binding sites and that the N-terminal half of Rev's Arginine-Rich Motif (ARM) is a primary ImpB binding epitope. Small-angle X-ray scattering (SAXS), crosslinking mass spectrometry and compensatory mutagenesis data suggest a structural model in which one Rev monomer binds to the C-terminal half of ImpB with Rev helix alpha-2 roughly parallel to the HEAT-repeat superhelical axis while the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by ImpB and highlight an atypical binding behaviour that distinguishes Rev from canonical cellular ImpB cargos.

The fate of Hepatitis E virus capsid protein is regulated by an Arginine-Rich Motif

Producing multifunctional proteins is one of the major strategies developed by viruses to condense their genetic information. Here, we investigated the molecular determinants of the multifunctionality of hepatitis E virus (HEV) ORF2 capsid protein. We previously identified 3 isoforms of ORF2 which are partitioned in different subcellular compartments to perform distinct functions. Notably, the infectious ORF2 (ORF2i) protein is the structural component of the virion, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. We identified a 5 amino acid Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region as a central regulator of the subcellular localizations and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation, promoting regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. Furthermore, the ARM likely serves as a cleavage site of the glycosylated ORF2 protein. Finally, it promotes ORF2 membrane association that is likely essential for particle assembly. In conclusion, our observations highlight ORF2 ARM as a unique central regulator of ORF2 addressing that finely controls the HEV lifecycle.

A structure-based mechanism for HEXIM displacement from 7SK

Productive transcriptional elongation of many cellular and viral mRNAs requires transcriptional factors to extract pTEFb from the 7SK snRNP by modulating the association between the HEXIM protein and the 7SK snRNA. Here we report the structure of the HEXIM arginine rich motif in complex with the apical stemloop-1 of 7SK (7SK-SL1apical) and detail how the HIV transcriptional regulator Tat from various subtypes overcome the structural constraints required to displace HEXIM. While the majority of interactions between 7SK and HEXIM and Tat are similar, critical differences exist that guide function. First, the conformational plasticity of 7SK enables the formation of three different base pair configurations at a critical remodeling site, which allows for the modulation required for HEXIM binding and its subsequent displacement by Tat. Furthermore, the specific sequence variations observed in various Tat subtypes all converge on remodeling 7SK at this region. Second, we show that HEXIM primes its own displacement by causing specific local destabilization upon binding — a feature that is then exploited by Tat to bind 7SK more efficiently. Overall, our study details the molecular environment presented by HEXIM and uncovers a destabilization-driven displacement strategy that increases the conformational sampling of 7SK-snRNP, which may allow diverse transcriptional factors to competitively regulate pTEFb.

Reporting Two SARS-CoV-2 Strains Based on A Unique Trinucleotide-Bloc Mutation and Their Potential Pathogenic Difference

SARS-CoV-2, the novel coronavirus behind COVID-19 pandemic is acquiring new mutations in its genome. Although some mutations provide benefits to the virus against human immune response, a number of them may result in their reduced pathogenicity and virulence. By analyzing more than 3000 high-coverage, complete genome sequences deposited in the GISAID database, here I report a unique 28881-28883:GGG>AAC trinucleotide-bloc mutation in the SARS-CoV-2 genome that results in two sub-strains, described here as SARS-CoV-2g (28881-28883:GGG genotype) and SARS-CoV-2a (28881-28883:AAC genotype). Computational analysis and literature review suggest that this bloc mutation would bring 203-204:RG(arginine-glycine)>KR(lysine-arginine) amino acid changes in the nucleocapsid (N) protein affecting the SR (serine-arginine)-rich motif of the protein, a critical region for the transcription of viral RNA and replication of the virus. Thus, 28881-28883:GGG>AAC bloc-mutation is expected to modulate the pathogenicity of the SARS-CoV-2. Remarkably, SARS-CoV-2g and SARS-CoV-2a strains can be linked with the heterogeneity of COVID-19 cases across different regions within and between countries by analyzing existing data. Sequence analysis suggests that severely affected cities, such as Milan, Lombardy, New York, Paris have the predominant presence of SARS-CoV-2g strains, whereas less affected places like Abruzzo, Lyon, Valencia have a relatively higher presence of SARS-CoV-2a, an indication that the latter strain may contribute to the reduced cases of COVID-19. A similar relationship is observed when Netherlands, Portugal are compared with Spain, France and Germany. These analyses suggest that the SARS-CoV-2 has already evolved into a less infective SARS-CoV-2a affecting COVID-19 cases in different regions. The time a country or region needs to acquire SARS-CoV-2a strains may be indicative to the time it would need to overcome the peak of the COVID-19 cases. To confirm these assumptions, prompt retrospective and prospective epidemiological studies should be conducted in different countries to understand the course of pathogenicity of the SARS-CoV-2a and SARS-CoV-2g. Potential drugs can be designed targeting 28881-28883 region of the N protein to modulate virus pathogenicity.

The Capsid Protein of Hepatitis E Virus Inhibits Interferon Induction via Its N-Terminal Arginine-Rich Motif

Hepatitis E virus (HEV) causes predominantly acute and self-limiting hepatitis. However, in HEV-infected pregnant women, the case fatality rate because of fulminant hepatitis can be up to 30%. HEV infection is zoonotic for some genotypes. The HEV genome contains three open reading frames: ORF1 encodes the non-structural polyprotein involved in viral RNA replication; ORF2 encodes the capsid protein; ORF3 encodes a small multifunctional protein. Interferons (IFNs) play a significant role in the early stage of the host antiviral response. In this study, we discovered that the capsid protein antagonizes IFN induction. Mechanistically, the capsid protein blocked the phosphorylation of IFN regulatory factor 3 (IRF3) via interaction with the multiprotein complex consisting of mitochondrial antiviral-signaling protein (MAVS), TANK-binding kinase 1 (TBK1), and IRF3. The N-terminal domain of the capsid protein was found to be responsible for the inhibition of IRF3 activation. Further study showed that the arginine-rich-motif in the N-terminal domain is indispensable for the inhibition as mutations of any of the arginine residues abolished the blockage of IRF3 phosphorylation. These results provide further insight into HEV interference with the host innate immunity.

Structural Basis of H2B Ubiquitination-Dependent H3K4 Methylation by COMPASS

SUMMARYThe COMPASS complex represents the prototype of the SET1/MLL family of methyltransferases that controls gene transcription by H3K4 methylation (H3K4me). Although H2B monoubiquitination (H2Bub) is well-known as a prerequisite histone mark for COMPASS activity, how the H2Bub-H3K4me crosstalk is catalyzed by COMPASS remains unclear. Here, we report the cryo-EM structures of an extended COMPASS catalytic module (CM) bound to the H2Bub and free nucleosome. The COMPASS CM clamps onto the nucleosome disk-face via an extensive interface to capture the flexible H3 N-terminal tail. The interface also sandwiches a critical Set1 arginine-rich motif (ARM) that auto-inhibits COMPASS. Unexpectedly, without enhancing COMPASS-nucleosome interaction, H2Bub activates the enzymatic assembly by packing against Swd1 and alleviating the inhibitory effect of the Set1 ARM upon fastening it to the acidic patch. By unmasking the spatial configuration of the COMPASS-H2Bub-nucleosome assembly, our studies establish the structural framework for understanding the long-studied H2Bub-H3K4me histone modification crosstalk.

Structural and Functional Characterization of RNA-binding Site of Rev and Rev-Response-Element RNA Complex via All Atom Molecular Dynamics Simulations

Nuclear export of viral mRNAs, is an essential step in the HIV replication cycle. This role is played by a small regulatory protein of HIV-1 called Rev.The N-terminal region of Rev contains an arginine-rich sequence. The arginine-rich motif (ARM) is located between amino acids 38-50 and forms an alpha-helical secondary structure. Expression of the structural proteins of human immunodeficiency virus type 1 requires the direct interaction of multiple copies of the viral Rev protein with its highly structured RNA target sequence, the Rev Response element (RRE). The major viral proteins are not produced if this transport of RNA is stopped. Therefore, knowledge of Rev structure is essential for understanding of its cooperative binding to the RRE, for understanding the mechanism of HIV infection and for the development of antiviral drugs that interfere with Rev’s essential functions and for acknowledgment of good candidate drugs for treatment of AIDS. To understand how REV interact with RRE element of HIV-RNA and its formation of oligomeric complex it is better to characterize the domain wise structure of REV with regard in function of each domain. Due to lack of structural data on Rev no single compound is reported as inhibitor of REV expect antiviral drugs. Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev Protein is much more important. The ARM is a highly specific sequence which allows for the multimerization of Rev and also binding of REV with RNA. Here we are first time exploring the structural characteristics of REV protein both in free form and in complex with RNA at domain function level especially explore the role of ARM motif in REV HIV-1 protein as RNA binding sites by molecular dynamics (MD) simulation and homology modeling studies. Results indicate that the arginine-rich motif (ARM) is crucial in stability of this complex. The residues ARG38, 39, 41, 43, 44, 48, 50, and ASN40 are most interacting with nucleobases of RRE in Crystal structure of Rev and Rev-response-element RNA complex. Our study plays a major role in elaboration of binding of RNA with REV and pave the way for further investigation for therapeutically agent for HIV.